1. Field of the Invention
The invention relates to a mammalian cell lacking type-1 parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH1R) activity and containing carboxyl-terminal parathyroid hormone receptor (CPTHR) activity. The invention also relates to a method of screening for agonists or antagonists for CPTHR.
2. Related Art
Parathyroid hormone (PTH) is the major physiologic regulator of blood calcium and phosphate, and it exerts potent effects upon cells in bone and cartilage (Heersche, J. N. M., et al., “Cellular Actions of Parathyroid Hormone on Osteoblasts and Osteoclasts Differentiation,” in The Parathyroids Basic and Clinical Concepts, Bilezikian, J. P. et al., (eds.), Raven Press, New York, N.Y., pp. 83-91 (1994)). PTH and PTH-related peptide (PTHrP) activate a common G protein-coupled receptor, the type-1 PTH/PTHrP receptor (PTH1R) (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. USA 89:2732-2736 (1992); Juppner, H., et al., Science 254:1024-1026 (1991); Juppner, H., Bone 25:87-90 (1999)). The PTH1R recognizes the highly conserved amino (N)-terminal domain of PTH (and the homologous N-terminus of PTHrP) and thus is fully activated by both PTH(1-34) and the intact hormone, PTH(1-84). Carboxyl(C)-fragments of intact PTH(1-84), such as PTH(39-84) or PTH(53-84), neither bind nor activate the PTH1R(Pines, M., et al., Endocrinology 135:1713-1716 (1994); Inomata, N., et al., Endocrinology 136:4732-4740 (1995); Bringhurst, F. R., et al., Am. J. Physiol. 255:E886-E893 (1988)). On the other hand, a possible physiologic role for this region of the hormone is indicated by observations that the amino acid sequence of the PTH C-terminal domain is highly homologous across species (Kbosla, S., et al., J. Bone Miner. Res. 3:689-698 (1988)); that PTH C-fragments, arising via both secretion from the parathyroid glands and proteolysis of PTH(1-84) in peripheral tissues, circulate in blood at levels much higher than those of the intact hormone; and that parathyroid secretion of PTH C-fragments is strongly regulated by serum calcium (D'Amour, P., et al., Am. J. Physiol. 251:E680-E687 (1986); Bringhurst, F. R., et al., J. Endocrinol. 122:237-245 (1989); Hanley, D. A., et al., J. Clin. Invest. 62:1247-1254 (1978); Martin, K., et al., J. Clin. Invest. 58:781-788 (1976); Dambacher, M. A., et al., Clinical Sci. 57:435-443 (1979)).
Studies with rat osteoblastic cell lines have shown that fragments from within the sequence PTH(35-84), which cannot activate PTH1Rs, regulate expression of alkaline phosphatase, osteocalcin, collagen α1 (I) and IGF binding protein-5 (Murray, T. M., et al., Calcified Tissue International 49:120-123 (1991); Nasu, M., et al., Endocrine J. 45:229-234 (1998); Takasu, H., et al., Endocrinology 137:5537-5543 (1996)). Others have found direct effects of PTH C-fragments upon osteoclasts and osteoclast progenitors (Kaji, H., et al., Endocrinology 136:842-848 (1995)) and upon expression of collagen α1 (I) and α1 (X) expression in hypertrophic chondrocytes (Erdmamu, S., et al., J. Cell Biol. 135:1179-1191 (1996)). Several such fragments also were shown to induce cytosolic free calcium transients in human fetal hypertrophic chondrocytes (Erdmamn, S., et al., Cell Calcium 23:413-421 (1998)). Direct physical evidence of a putative receptor with binding specificity for C-terminal PTH sequences (CPTHR) was obtained by crosslinking of the peptide 125I-[Tyr34]hPTH(19-84) (which does not bind to PTH1Rs) to 40 kD and 90 kD proteins in ROS 17/2.8 rat osteoblastic cells (Inomata, N., et al., Endocrinology 136:4732-4740 (1995); Takasu, H., et al., Endocrinology 137:5537-5543 (1996)).
Collectively, these observations indicate that CPTHRs are expressed normally in bone and cartilage and that they may be involved in physiologic control of cell differentiation and function in these tissues. The problem of renal osteodystrophy is of particular interest in this regard, as PTH C-fragments normally are cleared mainly by the kidneys and thus accumulate to very high levels in blood during renal failure (Bringhurst, F. R., et al., Am. J. Physiol. 255:ES86-E893 (1988); D'Amour, P., et al., Am. J. Physiol. 251:E680-E687 (1986); Martin, K., et al., J. Clin. Invest. 58:781-788 (1976); Dambacher, M. A., et al., Clinical Sci. 57:435-443 (1979)). Moreover hPTH(7-84) potently reduces the calcemic action of intact PTH(1-84) in a manner inconsistent with its low binding affinity to the PTH1R (Slatopolsky, E., et al., “A Novel Mechanism for Skeletal Resistance in Uremia,” in Program of the American Society of Nephrology Annual Meeting, Miami, Fla. (Abstract) (1999)).